Excipients for use in adeno-associated virus pharmaceutical formulations, and pharmaceutical formulations made therewith

ABSTRACT

Stable pharmaceutical compositions comprising recombinant adeno-associated virus (AAV) virions are described. The compositions provide protection against loss of recombinant AAV vector genomes and transduceability under conditions such as exposure to cycles of freezing and thawing and storage in glass or polypropylene vials. The compositions comprise recombinant AAV virions in combination with one or more dihydric or polyhydric alcohols, and, optionally, a detergent, such as a sorbitan ester. Also described are methods of using the compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 13/506,638, filedMay 4, 2012, now U.S. Pat. No. 8,852,607, which is a continuation ofU.S. Ser. No. 12/583,679, filed Aug. 24, 2009, now U.S. Pat. No.8,192,975, which is a continuation of U.S. Ser. No. 10/862,036, filedJun. 4, 2004, now U.S. Pat. No. 7,598,070, which is a continuation ofU.S. Ser. No. 10/340,389, filed Jan. 10, 2003, now U.S. Pat. No.6,764,845, which is a divisional application of U.S. Ser. No.09/453,317, filed Dec. 2, 1999, now U.S. Pat. No. 6,759,050, from whichapplications priority is claimed pursuant to 35 U.S.C. §120. U.S. Ser.No. 09/453,317 claims the benefit under 35 U.S.C. §119(e)(1) toprovisional patent application Ser. No. 60/110,689, filed Dec. 3, 1998.The above applications are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The present invention relates generally to DNA delivery methods. Moreparticularly, the invention relates to stable pharmaceuticalformulations comprising recombinant adeno-associated virus (rAAV)virions that provide protection against loss of transduceability due tomanipulation, storage, transport, and the like, of the formulation.

BACKGROUND

The commercialization of any chemical compound for use as apharmaceutical agent requires careful consideration of the formulationin which the chemical compound will be prepared, packaged and stored.The formulation must, of course, be compatible with human and/orveterinary administration. The formulation must be such that the agentretains potency for an extended period of time. Indeed, the formulationitself must be stable over a long period of time. The formulation mustbe compatible with techniques used for its purification, as well as forthe purification of the agent contained within the formulation.Ultimately, the formulation must be compatible with the material inwhich the agent will be stored. If the agent must be frozen forstability, it is preferable that the formulation provide some protectionagainst inactivation or denaturation due to freeze-thaw. In addition,the formulation should provide a suitable milieu for various dilutionsof the agent.

Typically, pharmaceutical agents are stored as lyophilized formulationsin a sterile container. A pharmaceutical agent formulation may belyophilized if it is stable in such a nonaqueous state. This is ofparticular importance if the formulation must be stored frozen, aslyophilization minimizes the deleterious sequelae that may occur when anaqueous preparation is frozen and subsequently thawed. A glass vial istypically used because of the compatibility of glass with presently usedsterilization techniques.

Adeno-associated virus (AAV) is a virus that readily transduces manyhuman tissue and cell types. Accordingly, AAV has been used for genetherapy and nucleic acid immunization. The use of AAV in these contextsrequires consideration of the above pharmaceutical formulationrequirements. For example, it would be preferred that an AAV-containingsample not be lyophilized because of the possibility that small amountsof virus could become aerosolized and inadvertently transduce anunintended host. However, because AAV is known to be stable under avariety of conditions that would inactivate most viruses, particularlyenveloped viruses, it was not previously believed that the preparationof AAV formulations would be problematic.

It was unexpected, therefore, to find that the activity of recombinantAAV (AAV) virions dropped significantly depending on the formulationused for storage and the conditions to which the formulation wasexposed. It has been found, for example, that the transduction activityof a rAAV formulation may depend on the nature of the container, theconstituents of the formulation, the temperature of the formulation, aswell as changes in temperature, and the concentration of the rAAVvirions stored.

It would, therefore, be a significant advancement in the art to provideformulations for storing rAAV virions which would preserve the activityof the rAAV virions for extended periods of times in containers made ofvarious materials, including glass.

DISCLOSURE OF THE INVENTION

The present invention is based on the discovery that Various excipientcompositions have a stabilizing effect on recombinant AAV virions, suchthat less rAAV vector genomes are lost and higher transduceabilitylevels are achieved as compared with AAV compositions that lack theexcipients described herein. Various forms of the different embodimentsdescribed herein can be combined.

In one embodiment, then, a pharmaceutical composition comprising rAAVvirions is provided. The composition provides protection against loss ofrAAV vector genomes and transduceability under conditions such asexposure to cycles of freezing and thawing and storage in glass orpolypropylene vials. The composition comprises a dihydric or polyhydricalcohol, such as one or more of sorbitol, polyethylene glycol, propyleneglycol, and, optionally, a detergent, such as a sorbitan ester.

In an additional embodiment, the pharmaceutical composition comprisesrAAV virions in an amount sufficient to provide a therapeutic effectwhen given in one or more doses and sorbitol present at a concentrationof about 1 wt. % to about 5 wt. % and a detergent present at aconcentration of about 0.1 wt. % to about 1 wt. %, wherein the detergentis polyoxyethylenesorbitan monolaurate (TWEEN-20) orpolyoxyethylenesorbitan monooleate (TWEEN-80).

In yet other embodiments, a method for protecting a recombinant AAVvirion from loss of activity resulting from exposure of the vision to acycle of freezing and thawing, is provided, as is a method forprotecting a recombinant AAV virion from loss of activity resulting fromstorage of the virion in a glass vessel. The methods comprise admixingthe virion with a virion-stabilizing composition comprising a dihydricor polyhydric alcohol. In particular embodiments, the alcohol is one ormore alcohols selected from the group consisting of polyethylene glycol,propylene glycol and sorbitol. The compositions used in the methodsoptionally include a detergent, such as a sorbitan ester.

In particular embodiments, the compositions used in the methods comprisesorbitol and a sorbitan ester selected from the group consisting ofpolyoxyethylenesorbitan monolaurate (TWEEN-20) andpolyoxyethylenesorbitan monooleate (TWEEN-80).

These and other embodiments of the subject invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is a bar graph illustrating the effect of temperature, vectordilution and storage in a glass vial on recombinant AAV vector(rAAV-hFIX) transduceability, as described in the examples. The numberson the x axis represent the storage temperature; ppvial representssamples stored in a polypropylene vial and gl vial represents samplesstored in glass.

FIG. 2 depicts the results of experiments conducted as described inExample 3 in which 293-HEK cells were transduced with 1×10⁸ vectorgenomes using samples stored in polypropylene or glass, in 1% or 5%sorbitol, at various dilutions as specified. The bars represent ng/ml ofrAAV-hFIX and the line graph above the bars represents data normalizedfor dilution.

FIG. 3 depicts the results of experiments conducted as described inExample 3 in which 293-HEK cells were transduced with 5×10⁸ vectorgenomes using samples stored in polypropylene or glass, in 1% or 5%sorbitol, at various dilutions as specified. The bars represent ng/ml ofrAAV-hFIX and the line graph above the bars represents data normalizedfor dilution.

FIG. 4 depicts the results of experiments conducted at −80° C. orambient (room) temperature for vector genome count and transduceabilityassay in which transduction was done using 1×10⁸ vector genomes asdescribed in Example 3. Maroon bars represent results of experimentsdone at room temperature and light blue bars represent experiments doneat −80° C.

FIG. 5 depicts the results of experiments conducted at −80° C. orambient (room) temperature for vector genome count and transduceabilityassay in which transduction was done using 5×10⁸ vector genomes asdescribed in Example 3. Maroon bars represent results of experimentsdone at room temperature and light blue bars represent experiments doneat −80° C.

FIG. 6 depicts the results obtained in the experiment described inExample 4 using the parameters given in Table 4. Green bars representexperiments conducted using 1×10⁸ vector genomes; red bars representexperiments conducted using 5×10⁸ vector genomes; pink bars representexperiments conducted using 1×10⁹ vector genomes; dark blue barsrepresent experiments conducted using 5×10⁹ vector genomes.

FIG. 7 depicts the results obtained in the experiment described inExample 4 using the parameters given in Table 4 and regraphed as vectorgenomes. Dark blue bars represent experiments conducted using media asthe diluent; pink bars represent experiments conducted using 0.1%TWEEN-20 as the diluent; yellow bars represent experiments conductedusing 0.2% TWEEN-20 as the diluent; red bars represent experimentsconducted using 0.5% TWEEN-20 as the diluent; green bars representexperiments conducted using 0.1% TWEEN-80 as the diluent; brown barsrepresent experiments conducted using 0.2% TWEEN-80 as the diluent;lavender bars represent experiments conducted using 0.5% TWEEN-80 as thediluent; teal blue bars represent experiments conducted using 2%PEG-3350 as the diluent; turquoise blue bars represent experimentsconducted using 3% PEG-3350 as the diluent; purple-striped barsrepresent experiments conducted using 2.25% glycine as the diluent;light blue bars represent experiments conducted using 0.1% TWEEN-20+2%PEG-3350+2.25% glycine as the diluent; blue bars represent experimentsconducted using 0.1% TWEEN-80+2% PEG-3350+2.25% glycine as the diluent.In all cases, the excipient included 1% sorbitol.

FIG. 8 depicts the results obtained in the experiment described inExample 5 to determine the effect of formulation composition on thestability of recombinant AAV vectors using the parameters described inTable 5. Light blue bars represent experiments conducted using media asthe diluent; yellow bars represent experiments conducted using 10%propylene glycol as the diluent; pink bars represent experimentsconducted using 25% propylene glycol as the diluent; turquoise blue barsrepresent experiments conducted using 50% propylene glycol as thediluent; dark blue bars represent experiments conducted using 18%PEG-400 as the diluent; light brown bars represent experiments conductedusing 25% propylene glycol+0.2% TWEEN-20 as the diluent; blue barsrepresent experiments conducted using 25% propylene glycol+0.2% TWEEN-80as the diluent.

FIG. 9 depicts the results obtained in the experiment described inExample 6 to determine the effect of various excipients and storageconditions on the stability of recombinant AAV vectors, as described inTable 6. Light blue bars represent experiments conducted using media asthe diluent and storage in glass at −80° C.; red bars representexperiments conducted using 0.5% TWEEN-80 as the diluent and storage inglass at −80° C.; green-striped bars represent experiments conductedusing 1% sorbitol as the diluent and storage in glass at −80° C.; yellowbars represent experiments conducted using media as the diluent andstorage in polypropylene at −80° C.; gray bars represent experimentsconducted using 0.5% TWEEN-80 as the diluent and storage inpolypropylene at −80° C.; purple-striped bars represent experimentsconducted using 1% sorbitol as the diluent and storage in polypropyleneat −80° C.; dark blue bars represent experiments conducted using mediaas the diluent and storage in glass at +4° C.; blue bars representexperiments conducted using 0.5% TWEEN-80 as the diluent and storage inglass at +4° C.; blue-striped bars represent experiments conducted using1% sorbitol as the diluent and storage in glass at +4° C.; mustardyellow bars represent experiments conducted using media as the diluentand storage in polypropylene at +4° C.; light yellow bars representexperiments conducted using 0.5% TWEEN-80 as the diluent and storage inpolypropylene at +4° C.; pink-striped bars represent experimentsconducted using 1% sorbitol as the diluent and storage in polypropyleneat +4° C.

FIG. 10 depicts the results obtained in the experiment described inExample 7 to determine the effect of various excipients on loss of rAAVvector activity in samples stored in glass or polypropylene vials.Lavender bars represent experiments conducted using media as the diluentand storage in glass at −80° C.; light blue-striped bars representexperiments conducted using 1% sorbitol as the diluent and storage inglass at −80° C.; dark blue-striped bars represent experiments conductedusing 10% propylene glycol (PG) as diluent and storage in glass at −80°C.; gray bars represent experiments conducted using 25% PG as diluentand storage in glass at −80° C.; blue bars represent experimentsconducted using 10% PG+0.2% TWEEN-80 as diluent and storage in glass at−80° C.; yellow bars represent experiments conducted using 25% PG+0.2%TWEEN-80 as diluent and storage in glass at −80° C.; brown-striped barsrepresent experiments conducted using 10% PG+0.5% TWEEN-80 as diluentand storage in glass at −80° C.; purple-striped bars representexperiments conducted using 25% PG+0.5% TWEEN-80 as diluent and storagein glass at −80° C.; dark purple bars represent experiments conductedusing media as the diluent and storage in polypropylene at +4° C.; pinkbars represent experiments conducted using 1% sorbitol as the diluentand storage in polypropylene at +4° C.; light yellow bars representexperiments conducted using 10% PG as diluent and storage inpolypropylene at +4° C.; turquoise blue bars represent experimentsconducted using 25% PG as diluent and storage in polypropylene at +4°C.; dark green bars represent experiments conducted using 10% PG+0.2%TWEEN-80 as diluent and storage in polypropylene at +4° C.; light bluebars represent experiments conducted using 25% PG+0.2% TWEEN-80 asdiluent and storage in polypropylene at +4° C.; green bars representexperiments conducted using 10% PG+0.5% TWEEN-80 as diluent and storagein polypropylene at +40° C.; dark blue bars represent experimentsconducted using 25% PG+0.5% TWEEN-80 as diluent and storage inpolypropylene at +4° C. The number “118” in the figure represents aspecific experiment number.

FIG. 11 depicts the results obtained in the experiment described inExample 8 to determine the effect of 5% sorbitol, alone and incombination with various excipients on the stability of recombinant AAVvectors after a freeze/thaw cycle of samples stored in a glass vial or apolypropylene tube. Light blue bars represent experiments conductedusing media as the diluent (without sorbitol) and storage in glass at−80° C.; pink bars represent experiments conducted using 5% sorbitol andstorage in glass at −80° C.; yellow bars represent experiments conductedusing 5% sorbitol+0.1% TWEEN-80 and storage in glass at −80° C.; greenbars represent experiments' conducted using 5% sorbitol+0.25% TWEEN-80and storage in glass at −80° C.; light brown bars represent experimentsconducted using 5% sorbitol+0.5% TWEEN-80 and storage in glass at −80°C.; turquoise blue bars represent experiments conducted using media asthe diluent (without soibitol) and storage in polypropylene at −80° C.;gray bats represent experiments conducted using 5% sorbitol and storagein polypropylene at −80° C.; blue bars represent experiments conductedusing 5% sorbitol+0.1% TWEEN-80 and storage in polypropylene at −80° C.;lime green bars represent experiments conducted using 5% sorbitol+0.25%TWEEN-80 and storage in polypropylene at −80° C.; red bars representexperiments conducted using 5% sorbitol+0.5% TWEEN-80 and storage inpolypropylene at −80° C.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, Vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, vol. I.& II (P. Tijssen, ed.);Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M.Knipe, eds.); Freshney Culture of Animal Cells, A Manual of BasicTechnique (Wiley-Liss, Third Edition); and Ausubel et al. (1991) CurrentProtocols in Molecular Biology (Wiley Inter science, NY).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

A. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

By “vector” is meant any genetic element, such ifs a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

By “AAV vector” is meant a vector derived from an adeno-associated virusserotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAV-6, etc. AAV vectors can have one or more of the AAV wild-typegenes deleted in whole or part, preferably the rep and/or cap genes(described below), but retain functional flanking ITR sequences (alsodescribed below). Functional ITR sequences are necessary for the rescue,replication and packaging of the AAV virion. Thus, an AAV vector isdefined herein to include at least those sequences required in cis forreplication and packaging (e.g., functional ITRs) of the virus. The ITRsneed not be the wild-type nucleotide sequences, and may be altered,e.g., by the insertion, deletion or substitution of nucleotides, so longas the sequences provide for functional rescue, replication andpackaging.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

By “AAV virion” is meant a complete virus particle, such as a wild-type(wt) AAV virus particle (comprising a linear, single-stranded AAVnucleic acid genome associated with an AAV capsid protein coat). In thisregard, single-stranded AAV nucleic acid molecules of eithercomplementary sense, e.g., “sense” or “antisense” strands, can bepackaged into any one AAV virion and both strands are equallyinfectious.

A “recombinant AAV virion,” or “rAAV virion” is defined herein as aninfectious, replication-defective virus composed of an AAV proteinshell, encapsidating a DNA molecule of interest which is flanked on bothsides by AAV ITRs. An rAAV virion is produced in a suitable host cellwhich has had an AAV vector, AAV helper functions and accessoryfunctions introduced therein. In this manner, the host cell is renderedcapable of encoding AAV polypeptides that are requited for packaging theAAV vector (containing a recombinant nucleotide sequence of interest)into recombinant virion particles for subsequent gene delivery.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare known in the art. See, e.g., Graham et al. (1973) Virology, 52:456,Sambrook et al. (1989) Molecular Cloning, a laboratory manual, ColdSpring Harbor Laboratories, New York, Davis et al. (1986) Basic Methodsin Molecular Biology, Elsevier, and Chu et al. (1981) Gene 1:197. Suchtechniques can be used to introduce one or more exogenous DNA moieties,such as a plasmid vector and other nucleic acid molecules, into suitablehost cells. The term refers to both stable and transient uptake of thegenetic material.

The term “transduction” denotes the delivery of a DNA molecule to arecipient cell either in vivo or in vitro, via a replication-defectiveviral vector, such as via a recombinant AAV virion.

By “DNA” is meant a polymeric form of deoxyribonucleotides (adenine,guanine, thymine, or cytosine) in double-stranded or single-strandedform, either relaxed and supercoiled. This term refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includes single- anddouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having the sequence homologous to the mRNA). Theterm captures molecules that include the four bases adenine, guanine,thymine, or cytosine, as well as molecules that include base analogswhich are known in the art.

A “gene” or “coding sequence” or a sequence which “encodes” a particularprotein, is a nucleic acid molecule which is transcribed (in the case ofDNA) and translated (in the case of mRNA) into a polypeptide in vitro orin vivo when placed under the control of appropriate regulatorysequences. The boundaries of the gene are determined by a start codon atthe 5′ terminus (corresponding to the amino terminal of the encodedprotein) and a translation stop codon at the 3′ (corresponding to thecarboxy terminal of the encoded protein) terminus. A gene can include,but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomicDNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the gene sequence.

The term “control elements” refers collectively to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which collectively provide forthe replication, transcription and translation of a coding sequence in arecipient cell. Not all of these control elements need always be presentso long as the selected coding sequence is capable of being replicated,transcribed and translated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene that is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence and canbe on the same (cis) or different (trans) nucleic acid molecule from thecoding sequence, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3′” or “5′” relative toanother sequence, it is to be understood that it is the position of thesequences in the “sense” or “coding” strand of a DNA molecule that isbeing referred to as is conventional in the art.

By “polyhydric alcohol” is meant an alcohol containing three or morehydroxyl groups. Generally, alcohols having three hydroxyl groups(trihydric) are glycerols, while those with more than three hydroxylgroups are sugar alcohols. A “dihydric alcohol” is one having twohydroxyl groups. Examples of polyhydric and dihydric alcohols are givenbelow.

B. GENERAL METHODS

The present invention provides stable pharmaceutical compositionscomprising rAAV virions. The compositions remain stable and active evenwhen subjected to freeze/thaw cycling and when stored in containers madeof various materials, including glass.

Recombinant AAV virions containing a heterologous nucleotide sequence ofinterest can be used for gene delivery, such as in gene therapyapplications, for the production of transgenic animals, in nucleic acidvaccination, ribozyme and antisense therapy, as well as for the deliveryof genes in vitro, to a variety of cell types.

Generally, rAAV virions are introduced into the cells of a subject usingeither in vivo or in vitro transduction techniques. If transduced invitro, the desired recipient cell will be removed from the subject,transduced with rAAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic cells can be used where thosecells will not generate an inappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant rAAV virions with the cells e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest using conventional techniques such as Southern blots and/orPCR, or by using selectable markers. Transduced cells can then beformulated into pharmaceutical compositions, described more fully below,and the composition introduced into the subject by various routes, suchas by intramuscular, intravenous, intra arterial, subcutaneous andintraperitoneal injection, or by injection into smooth muscle, usinge.g., a catheter, or directly into an organ.

For in vivo delivery, the rAAV virions will be formulated into apharmaceutical composition and will generally be administeredparenterally, e.g., by intramuscular injection directly into skeletalmuscle, intra articularly, intravenously or directly into an organ.

Appropriate doses will depend on the subject being treated (e.g., humanor nonhuman primate or other mammal), age and general condition of thesubject to be treated, the severity of the condition being treated, themode of administration of the rAAV virions, among other factors. Anappropriate effective amount can be readily determined by one of skillin the art.

Thus, a “therapeutically effective amount” will fall in a relativelybroad range that can be determined through clinical trials. For example,for in vivo injection, i.e., injection directly to the subject, atherapeutically effective dose will be on the order of from about 10⁵ to10¹⁶ of the rAAV virions, more preferably 10⁸ to 10¹⁴ rAAV virions. Forin vitro transduction, an effective amount of rAAV virions to bedelivered to cells will be on the order of 10⁵ to 10¹³, preferably 10⁸to 10¹³ of the rAAV virions. If the composition comprises transducedcells to be delivered back to the subject, the amount of transducedcells in the pharmaceutical compositions will be from about 10⁴ to 10¹⁰cells, more preferably 10⁵ to 10⁸ cells. The dose, of course, depends onthe efficiency of transduction, promoter strength, the stability of themessage and the protein encoded thereby, etc. Effective dosages can bereadily established by one of ordinary skill in the art through routinetrials establishing dose response curves.

Dosage treatment may be a single dose schedule or a multiple doseschedule to ultimately deliver the amount specified above. Moreover, thesubject may be administered as many doses as appropriate. Thus, thesubject may be given, e.g., 10⁵ to 10¹⁶ rAAV virions in a single dose,or two, four, five, six or more doses that collectively result indelivery of, e.g., 10⁵ to 10¹⁶ rAAV virions. One of skill in the art canreadily determine an appropriate number of doses to administer.

Pharmaceutical compositions will thus comprise sufficient geneticmaterial to produce a therapeutically effective amount of the protein ofinterest, i.e., an amount sufficient to reduce or ameliorate symptoms ofthe disease state in question or an amount sufficient to confer thedesired benefit. Thus, rAAV virions will be present in the subjectcompositions in an amount sufficient to provide a therapeutic effectwhen given in one or more doses. The rAAV virions can be provided aslyophilized preparations and diluted in the virion-stabilizingcompositions for immediate or future use. Alternatively, the rAAVvirions may be provided immediately after production and stored forfuture use.

The pharmaceutical compositions will also contain a pharmaceuticallyacceptable excipient. Such excipients include any pharmaceutical, agentthat does not itself induce the production of antibodies harmful to theindividual receiving the composition, and which may be administeredwithout undue toxicity. Pharmaceutically acceptable excipients include,but are not limited to, liquids such as water, saline, glycerol andethanol. Pharmaceutically acceptable salts can be included therein; forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles. A thorough discussion of pharmaceutically acceptableexcipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (MackPub. Co., N.J. 1991).

Preferred excipients confer a protective effect on the rAAV virion suchthat loss of rAAV virions, as well as transduceability resulting fromformulation procedures, packaging, storage, transport, and the like, isminimized. These excipient compositions are therefore considered“virion-stabilizing” in the sense that they provide higher rAAV viriontiters and higher transduceability levels than their non-protectedcounterparts, as measured using standard assays, such as the assaysdescribed in the experimental section. These compositions thereforedemonstrate “enhanced transduceability levels” as compared tocompositions lacking the particular excipients described herein, and aretherefore more stable than their non-protected counterparts.

Excipients that are used to protect the rAAV virion from activitydegradative conditions include, but are not limited to, detergents,proteins, e.g., ovalbumin and bovine serum albumin, amino acids, e.g.,glycine, polyhydric and dihydric alcohols, such as but not limited topolyethylene glycols (PEG) of varying molecular weights, such asPEG-200, PEG-400, PEG-600, PEG-1000, PEG-1450, PEG-3350, PEG-6000,PEG-8000 and any molecular weights in between these values, withmolecular weights of 1500 to 6000 preferred; propylene glycols (PG),sugar alcohols, such as a carbohydrate, preferably, sorbitol. Thedetergent, when present; can be an anionic, a cationic, a zwitterionicor a nonionic detergent. A preferred detergent is a nonionic detergent.More preferably, the nonionic detergent is a sorbitan ester, e.g.,polyoxyethylenesorbitan monolaurate (TWEEN-20) polyoxyethylenesorbitanmonopalmitate (TWEEN-40), polyoxyethylenesorbitan monostearate(TWEEN-60), polyoxyethylenesorbitan tristearate (TWEEN-65),polyoxyethylenesorbitan monooleate (TWEEN-80), polyoxyethylenesorbitantrioleate (TWEEN-85), preferably TWEEN-20 and/or TWEEN-80. Theseexcipients are commercially available from a number of vendors, such asSigma, St. Louis, Mo.

The amount of the various excipients present will vary and is readilydetermined by one of skill in the art. For example, a protein excipient,such as BSA, if present, will generally be present at a concentration ofbetween 1.0 wt. % to about 20 wt. %, preferably 10 wt. %. If an aminoacid such as glycine is used in the formulations, it will generally bepresent at a concentration of about 1 wt. % to about 5 wt. %. Acarbohydrate, such as sorbitol, if present, will be present at aconcentration of about 0.1 wt. % to about 10 wt. %, preferably betweenabout 0.5 wt. % to about 15 wt. %, more preferably about 1 wt. % toabout 5 wt. %. If PG is present, it will generally be present on theorder of about 2 wt. % to about 40 wt. %, preferably about 10 wt. % topabout 25 wt. %. If propylene glycol is used in the subject formulations,it will typically be present at a concentration of about 2 wt. % toabout 60 wt. %, preferably about 5 wt. % to about 30 wt. %. If adetergent such as a sorbitan ester (TWEEN) is present, it will generallybe present at a concentration of about 0.05 wt. % to about 5 wt. %,preferably between about 0.1 wt. % and about 1 wt. %.

In one-preferred embodiment, an aqueous virion-stabilizing formulationcomprises a carbohydrate, such as sorbitol, at a concentration ofbetween 0.1 wt. % to about 10 wt. %, preferably between about 1 wt. % toabout 5 wt. %, and a detergent, such as a sorbitan ester (TWEEN) at aconcentration of between about 0.05 wt. % and about 5 wt. %, preferablybetween about 0.1 wt. % and about 1 wt. %. Virions are generally presentin the composition in an amount sufficient to provide a therapeuticeffect when given in one or more doses, as defined above.

C. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

MATERIALS AND METHODS Production of Recombinant AAV Virions

Recombinant AAV virions can be produced using the method described incommonly owned U.S. Pat. No. 5,622,856 to Natsoulis, the disclosure ofwhich is incorporated herein by reference.

Briefly, the method includes the steps of: introducing an AAV vectorinto a suitable host cell; introducing an AAV helper construct into thehost cell to express essential AAV helper functions; expressing viralhelper functions in the host cell; and culturing the cell to producerAAV virions. The AAV vector and AAV helper constructs can betransfected into the host cell, either sequentially or simultaneously,using techniques known to those of skill in the art. The expression ofviral helper functions can be provided by infecting the host cell with asuitable helper virus selected from the group of adenoviruses,herpesviruses and vaccinia viruses. The viral helper functionstransactivate AAV promoters present in the AAV helper construct thatdirect the transcription and translation of AAV rep and cap regions.Thus, rAAV virions harboring a selected heterologous nucleotide sequenceare formed and can be purified from the preparation using known.

The supernatant obtained from the host cell is titered for rAAV viralproduction either by dot blot to calculate the number of viral genomesor by transducing cells with the rAAV thus produced and harvested, andassaying for β-galactosidase activity to determine functional units asindicating rAAV LacZ transduceability. Transducing vector titers can bedetermined by infecting 293 cells, or any cell competent fortransfection with AAV, with a dilution series of the rAAV virions. After24 hours, the cells are fixed and stained with X-Gal. Sanes et al.(1986) EMBO 5:3133-3142. The titer is calculated by quantifying thenumber of blue cells.

Construction of pAAVLacZ—

An AAV vector carrying the lacZ gene (pAAV-lacZ) was constructed asfollows. The AAV coding region of pSub201 (Samulski et al. (1987) J.Virol 61:3096-3101), between the XbaI sites, was replaced with EcoRIlinkers, resulting in plasmid pAS203. The EcoRI to HindIII fragment ofpCMVβ (CLONETECH) was rendered blunt ended and cloned in the Klenowtreated EcoRI site of pAS203 to yield pAAV-lacZ.

Example 1 Effect of Freezing/Thaw Cycle on Recombinant AAV Activity

This experiment was done to determine the effect of a freeze/thaw cycleon rAAV activity. As shown in the table, about 75% of the activity islost if no agent is added to the rAAV before it is frozen. The additionof bovine serum albumin (BSA) or polyoxyethylenesorbitan monolaurate(TWEEN-20) alone improved the recovery (about 50%). Sorbitol, however,completely protected the sample from freeze/thaw inactivation. Theseexperiments were performed in polypropylene vials.

AAV-lacZ was chromatographed on an ion exchange column. A small volumefrom each of the fractions from the column was assayed for blue cellactivity on the same day, prior to freezing the sample. The peakfraction contained 47% of the initial load on the column.

The remainder of each fraction was split into 4 portions and thefollowing excipients added to each portion:

Portion a—no excipient;

Portion b—BSA to a final concentration of 10%;

Portion c—sorbitol to a final concentration of 5%;

Portion d—TWEEN-20 to a final concentration of 0.5%;

These samples were frozen, thawed a few days later and assayed for bluecell activity. The results are summarized in Table 1

TABLE 1 Sample Excipient % Yield Active fraction, pre-freeze None 47%Active fraction, post freeze-thaw None 15% Active fraction, postfreeze-thaw BSA 27% Active fraction, post freeze-thaw Sorbitol 52%Active fraction, post freeze-thaw TWEEN-20 29%

These results indicate that a single freeze/thaw cycle can result in asignificant reduction in rAAV activity. This reduction in activity canbe abrogated by addition of protective agents such as proteins,polyhydric alcohols and detergents, all of which are believed to act bydifferent mechanisms. In this experiment, sorbitol had the greatestprotective effect; essentially no loss of activity following freezingand thawing was observed in the sorbitol-containing sample.

Example 2 Effect of Vector Dilution, Temperature and Storage in GlassVials on Recombinant AAV Activity

Initial stability experiments were conducted to determine the effect ofvector dilution, recovery from polypropylene (pp) vials and glass (gl)vials and the effect of temperature (−80° C., 2-8° C., 37° C.) onstorage stability and the effect of the addition of sorbitol onstability and recovery.

Samples of rAAV in a 1% sorbitol solution in phosphate buffered saline(PBS) were assayed undiluted as well as diluted 1-fold to about 10-fold(1.25 ml-12.5 ml) within 1% sorbitol/DPBS buffer. 0.5 ml aliquots ofeach member of the dilution series were placed in different storageconditions.

Following freezing, the samples were thawed and analyzed for vectorgenomes and for transduceability as described in Example 1. Fortransduceability studies, the vector genome titer used in the experimentwas calculated using the starting concentration of undiluted vector.Each sample wag diluted in complete Dulbecco's Modified Eagle Medium(DMEM) such that 1×10⁸ or 5×10⁸ vector genomes could be added to 298-HEKcells in a volume of 10 to 20 μL.

The stability-indicating assay used was loss of transduceability. Lossof transduceability was measured by human factor IX protein (hFIX)production following transduction with rAAV of 293 human embryonickidney (HEK) cells. In this assay, different amounts of rAAV-hFIX vectorwere added to HEK cells and the ability to transduce was measured. Thefactor IX protein produced and secreted as a consequence of infectionwas measured using an ELISA technique. The results are reported in ng/mLof human-factor IX protein (hFIX).

As can be seen by the results depicted in FIG. 1, storing rAAV in glassregardless of the temperature that it was stored at caused the rAAVactivity to drop. In this experiment it appears that activity andtemperature are inversely related—i.e., the lower the temperature thehigher the activity.

Example 3 Effect of Storage in Glass on Recombinant AAV Activity

The results of the experiment described in Example 0.2 indicate thatstorage of rAAV in glass resulted in a loss of activity. This experimentwas designed to examine whether this was due merely to the fact thatrAAV was adsorbed to the glass vial.

In order to rule out this possibility, the number of genomes (the numberof DNA molecules encapsulated in AAV as determined by Southern Blot dot)added to the glass and polypropylene vials was determined. The rAAV wasallowed to sit in the glass and polypropylene vials in variousformulations and temperatures. Two aliquots of rAAV virus were taken:the first used to recount the number of genomes recovered and the secondto determine activity (functional units as opposed to genomes) asdetermined in Example 1. The results indicate that when rAAV is storedin glass the activity drops significantly. This occurs at variousdilutions of rAAV.

rAAV samples were frozen undiluted as well as diluted 5-fold, 50-fold or100-fold. The diluent used was such that the final concentration ofsorbitol was either 5% or 1%. Duplicate samples were placed in glassvials and polypropylene tubes. Samples were placed at −80° C. or atambient temperature. Following freezing, the samples were thawed andanalyzed for vector genomes and for transduceability as described above.For transduceability studies, the vector genome titer used in theexperiment was calculated using the starting concentration of undilutedvector. Each sample was diluted in complete DMEM such that 1×10⁸ or5×10⁸ vector genomes could be added to 293-HEK cells in a volume of 10to 20 μL.

The stability-indicating assays used were loss of vector genomes andloss of transduceability. Loss of vector genomes was measured using adot blot assay. This assay involves extraction of vector DNA from thesample, denaturing the DNA, and loading it on a nylon membrane. Byhybridizing this DNA to a complimentary radioactive DNA probe, thenumber of vector genomes can be calculated by comparison to a standard.Loss of transduceability was measured as described in Example 2.

Table 2 lists the data obtained for 293-HEK cells transduced with 1×10⁸vector genomes. The data is shown for the samples incubated at −80° C.Column 2 shows the transduceability, column 3 is the measured vectorgenomes/ml and column 4 is the data in column 3 multiplied by thedilution factor. In the table, “ppS” indicates polypropylene stockcontainer and “ppC” indicates polypropylene container.

TABLE 2 3 4 2 Vector Normalized 1 ng/ml Genomes/ Vector Sample hFIX mlGenomes 5%, ppS, −80° C., stock 11.6 1.5 × 10¹² 1.5 × 10¹² 5%, ppC, −80°C., 1:5 6.6 2.3 × 10¹¹ 1.2 × 10¹² 5%, glass, −80° C., 1:5 3.8 1.9 × 10¹¹9.5 × 10¹¹ 1%, ppC, −80° C., 1:5 4.2 1.9 × 10¹¹ 9.5 × 10¹¹ 1%, glass,−80° C., 1:5 2.1 1.7 × 10¹¹ 8.5 × 10¹¹ 5%, ppC., −80° C., 1:50 5.9 2.5 ×10¹⁰ 1.3 × 10¹² 5%, glass, −80° C., 1:50 4.5 1.5 × 10¹⁰ 7.5 × 10¹¹ 1%,ppC, −80° C., 1:50 4.3 1.7 × 10¹⁰ 8.5 × 10¹¹ 1%, glass, −80° C., 1:504.6 1.9 × 10¹⁰ 9.5 × 10¹¹ 5%, ppC, −80° C., 1:100 5.5 1.5 × 10¹⁰ 1.5 ×10¹² 5%, glass, −80° C., 1:100 4.0 9.3 × 10⁹  9.3 × 10¹¹ 1%, ppC, −80°C., 1:100 4.6 1.2 × 10¹⁰ 1.2 × 10¹² 1%, glass, −80° C., 1:100 2.4 1.1 ×10¹⁰ 1.1 × 10¹²

FIG. 2 depicts the data for the vector genome and transduceabilityresults in which 1×10⁸ vector genomes were used for the transduceabilityassay. Vector genomes obtained experimentally have been normalized forthe dilution to compare with the transduceability results. In addition,the vector genome results have been divided by 1×10¹¹ to producemanageable numbers for the graph.

Table 3 and FIG. 3 depict the results of experiments conducted asdescribed above except that the transduceability assay for 293-HEK cellstransduced with 5×10⁸ vector

TABLE 3 3 4 2 Vector Normalized 1 ng/ml Genomes/ Vector Sample hFIX mlGenomes 5%, ppS, −80° C., stock 78.8 1.5 × 10¹² 1.5 × 10¹² 5%, ppC, −80°C., 1:5 48.3 2.3 × 10¹¹ 1.2 × 10¹² 5%, glass, −80° C., 1:5 37.1 1.9 ×10¹¹ 9.5 × 10¹¹ 1%, ppC, −80° C., 1:5 29.9 1.9 × 10¹¹ 9.5 × 10¹¹ 1%,glass, −80° C., 1:5 23.9 1.7 × 10¹¹ 8.5 × 10¹¹ 5%, ppC, −80° C., 1:5011.3 2.5 × 10¹⁰ 1.3 × 10¹² 5%, glass, −80° C., 1:50 18.5 1.5 × 10¹⁰ 7.5× 10¹¹ 1%, ppC, −80° C., 1:50 8.6 1.7 × 10¹⁰ 8.5 × 10¹¹ 1%, glass, −80°C.; 1:50 4.2 1.9 × 10¹⁰ 9.5 × 10¹¹ 5%, ppC, −80° C., 1:100 11.2 1.5 ×10¹⁰ 1.5 × 10¹² 5%, glass, −80° C., 1:100 30.7 9.3 × 10⁹  9.3 × 10¹¹ 1%,ppC, −80° C., 1:100 14.5 1.2 × 10¹⁰ 1.2 × 10¹² 1%, glass, −80° C., 1:1007.1 1.1 × 10¹⁰ 1.1 × 10¹²

FIG. 4 and FIG. 5 depict the results of experiments conducted at −80° C.or ambient temperature for vector genome count and transduceabilityassay in which transduceability was done using 1×10⁸ vector genomes(FIG. 4) and 5×10⁸ vector genomes (FIG. 5.) These results from thetransduceability experiments indicate that as the vector is diluted itdecreases in infective capability. It can also be seen that samplesprepared in 1% sorbitol have reduced transduceability compared to thoseprepared in 5% sorbitol. The results also indicate that storage in glassat −80° C. results in reduced transduceability compared topolypropylene.

Thus, it appears that there can be a physical loss of vector in thecontainer under the conditions examined in these experiments. However,from the vector genome data it can be observed that there is noobservable change in the number of vector genomes. The % correlation ofvariance (% CV) in the range of vector genomes (see Tables 2 and 3) is22.4, well within the variability of the dot blot assay. However, the %CV for the transduceability for both titers, i.e., 1×10⁸ vector genomesand 5×10⁸ vector genomes, is greater than the variability of thetransduceability assay (about 30%). Accordingly; it appears that, ratherthan a loss in vector genome number, the vector per se is losingtransduceability.

Example 4 The Effect of Added Excipients on the Stability of RecombinantAAV-I

These experiments were designed to study the effect of differentformulations containing 1% sorbitol and various concentrations ofTWEEN-20, TWEEN-80, polyethylene glycol (PEG), glycine and combinationsthereof. Virus placed in growth media was used as a baseline. The tubesused throughout were polypropylene. The samples were maintained in theformulations for 1 hr at room temperature before transducing culturecells.

The stability of the AAV vector was measured using the loss oftransduceability assay described in Example 2 to further explore theeffect of added excipients. Samples were diluted in media or in thebuffer excipient to give concentrations of 1×10⁸, 5×10⁸, 1×10⁹ or 5×10⁹in 15 μL. Samples were placed in polypropylene tubes for about 1 hourand then used to transduce 293-HEK cells.

The formulations of excipients and results are given in ng/ml of humanfactor IX in Table 4 and the results listed therein are depicted in FIG.6 and FIG. 7. In all cases, the excipient included 1% sorbitol.

TABLE 4 Diluent Concentration (All experimental excipients contained 1%Sorbitol) (ng/ml) Media 30.5 194.5 353.1 615.8  0.1% TWEEN-20 26.4 178.6347.7 616.9  0.2% TWEEN-20 31.8 180.6 384.4 623.9  0.5% TWEEN-20 30.2194.6 355.8 654.6  0.1% TWEEN-80 29.2 178.1 360.8 595.9  0.2% TWEEN-8035.6 186.4 399.8 669.0  0.5% TWEEN-80 29.8 183.6 344.3 588.6   2%PEG-3350 25.0 164.5 331.5 604.4   3% PEG-3350 28.6 162.0 354.9 556.02.25% glycine 14.9 115.6 240.8 516.3  0.1% TWEEN-20 + 2% PEG + 2.25%glycine 23.5 143.5 329.9 533.1  0.1% TWEEN-80 + 2% PEG + 2.25% glycine24.0 140.6 329.5 532.3 Particles/well 1 × 10⁸ 5 × 10⁸ 1 × 10⁹ 5 × 10⁹

These data indicate that the addition of TWEEN seems to have stabilizedrAAV, and PEG and glycine do little and may even reduce the overallactivity of rAAV.

Example 5 The Effect of Added Excipients on the Stability of RecombinantAAV-II

The stability of the AAV vector was measured as described in Example 4.The formulations of excipients and results are given in Table 5 and theresults listed therein are depicted in FIG. 8. In all cases, theexcipient included 1% sorbitol.

TABLE 5 Diluent Concentration All experimental excipients contained 1%Sorbitol (ng/ml) Media 28.5 192.2 269.2 607.0 10% Propylene Glycol 26.0144.7 313.3 607.0 25% Propylene Glycol 20.9 170.8 292.4 568.1 50%Propylene Glycol 26.9 163.4 256.5 600.1 18% PEG-400 14.8 94.2 224.7701.1 25% Propylene Glycol + 0.2% TWEEN-20 49.7 235.7 352.3 834.9 25%Propylene Glycol + 0.2% TWEEN-80 47.5 314.2 542.6 919.9 Particles/well 1× 10⁸ 5 × 10⁸ 1 × 10⁹ 5 × 10⁹

Example 6 The Effect of Added Excipients on the Stability of RecombinantAAV: Comparison of Glass and Polypropylene Vials

This experiment was designed to study the effect of 1% sorbitol andTWEEN-80 on the activity of rAAV stored in glass vials compared to theeffect on activity of rAAV stored in polypropylene vials at twotemperatures (4° C. and −80° C.).

The stability of the AAV vector was measured as described using 5×10⁷,1×10⁸ or 5×10⁸ particles/well. Each sample was done in a glass vial (GV)or a polypropylene tube (PT), and stored at room temperature (+4° C.) orat −80° C. overnight. The formulations of excipients and results aregiven in Table 6 and the results listed therein are depicted in FIG. 9.

TABLE 6 Diluent Concentration (ng/ml) Media, glass, −80° C. 23.6 42.8243.3 0.5% TWEEN-80, glass, −80° C. 23.5 46.7 254.7   1% sorbitol,glass, −80° C. 6.2 8.9 59.2 Media, polypropylene, −80° C. 26.4 48.1251.1 0.5% TWEEN-80, polypropylene, −80° C. 30.2 56.0 228.2   1%sorbitol, polypropylene, −80° C. 12.2 23.3 185.9 Media, glass, +4° C.19.7 34.9 247.1 0.5% TWEEN-80, glass, +4° C. 31.1 58.7 282.2   1%sorbitol, glass, +4° C. 7.0 14.5 111.9 Media, polypr4ylene, +4° C. 18.130.8 212.7 0.5% TWEEN-80, polypropylene, +4° C. 23.3 46.6 248.6   1%sorbitol, polypropylene, +4° C. 15.2 31.9 200.9 Particles/well 5 × 10⁷ 1× 10⁸ 5 × 10⁸

These data indicate that 1% sorbitol does not provide a significantprotective effect for rAAV activity when-stored in glass vials. Whensorbitol is used alone, it provides a protective effect on rAAV storedin polypropylene vials. There is also an apparent protective effect whenthe sample is stored at 4° C. rather than at −80° C. The inclusion ofTWEEN in the formulation reverses the reduced activity caused by storageof an rAAV sample in a glass vial.

Example 7 The Effect of Added Excipients on the Stability of RecombinantAAV-III: Comparison of Storage in Glass and Polypropylene Vials

The stability of the AAV vector was measured as described in Example 5.The formulations of excipients and results are given in FIG. 10. Thedata indicate that neither propylene glycol (PG) nor sorbitol aloneprotect against loss of activity of an rAAV sample stored in a glassvial. When PG and TWEEN were combined, loss of activity was minimizedand, in fact, it appears that PG and TWEEN together may have asynergistic effect on activity.

Example 8 The Effect of Added Excipients on the Stability of RecombinantAAV: the Effect of 5% Sorbitol

These experiments were designed to study the effect of 5% sorbitol, incombination with various concentrations of TWEEN on the activity ofrAAV.

The stability of the AAV vector after a freeze/thaw cycle was measuredas described in Example 6 using 1×10⁸, 5×10⁸ or 1×10⁹ particles/well.Each sample was done in a glass vial or a polypropylene tube, and storedat −80° C. overnight. The formulations of excipients and results aregiven in FIG. 11.

A formulation containing 5% sorbitol provided a partial protectiveeffect against loss of rAAV activity when the sample was stored in aglass vial. However, the addition of TWEEN provided a significantlygreater protective effect.

Thus, formulations for enhancing the stability of recombinant AAVpreparations are described. Although preferred embodiments of thesubject invention have been described in some detail, it is understoodthat obvious variations can be made without departing from the spiritand the scope of the invention as defined by the appended claims.

1. A pharmaceutical composition comprising recombinant adeno-associatedvirus (AAV) virions and at least one dihydric or polyhydric alcohol. 2.The pharmaceutical composition of claim 1, wherein the dihydric orpolyhydric alcohol is one or more alcohols selected from the groupconsisting of polyethylene glycol, propylene glycol and sorbitol.
 3. Thepharmaceutical composition of claim 2, wherein the one or more alcoholsis sorbitol and the sorbitol is present at a concentration of about 0.1wt. % to about 10 wt. %.
 4. The pharmaceutical composition of claim 3,wherein sorbitol is present at a concentration of about 1 wt. % to about5 wt. %.
 5. The pharmaceutical composition of claim 2, wherein the oneor more alcohols is polyethylene glycol and the polyethylene glycol ispresent at a concentration of about 2 wt. % to about 40 wt. %.
 6. Thepharmaceutical composition of claim 5, wherein polyethylene glycol ispresent at a concentration of about 10 wt. % to about 25 wt. %.
 7. Thepharmaceutical composition of claim 2, wherein the one or more alcoholsis propylene glycol and the propylene glycol is present at aconcentration of about 2 wt. % to about 60 wt. %.
 8. The pharmaceuticalcomposition of claim 7, wherein propylene glycol is present at aconcentration of about 5 wt. % to about 30 wt. %.
 9. The pharmaceuticalcomposition of claim 1, further comprising a detergent.
 10. Thepharmaceutical composition of claim 9, wherein the detergent is anonionic detergent.
 11. The pharmaceutical composition of claim 10,wherein the detergent is a sorbitan ester.
 12. The pharmaceuticalcomposition of claim 11, wherein the detergent is selected from thegroup consisting of polyoxyethylenesorbitan monolaurate (TWEEN-20),polyoxyethylenesorbitan monopalmitate (TWEEN-40),polyoxyethylenesorbitan monostearate (TWEEN-60), polyoxyethylenesorbitantristearate (TWEEN-65), polyoxyethylenesorbitan monooleate (TWEEN-80)and polyoxyethylenesorbitan trioleate (TWEEN-85).
 13. The pharmaceuticalcomposition of claim 12, wherein the detergent ispolyoxyethylenesorbitan monolaurate (TWEEN-20) present at aconcentration of about 0.05 wt. % to about 5 wt. %.
 14. Thepharmaceutical composition of claim 12, wherein the detergent ispolyoxyethylenesorbitan monooleate (TWEEN-80), present at aconcentration of about 0.05 wt. % to about 5 wt. %.
 15. A pharmaceuticalcomposition comprising recombinant AAV virions present in an amountsufficient to provide a therapeutic effect when given in one or moredoses, sorbitol present at a concentration of about 1 wt. % to about 5wt. % and a detergent present at a concentration of about 0.1 wt. % toabout 1 wt. %, wherein the detergent is polyoxyethylenesorbitanmonolaurate (TWEEN-20) or polyoxyethylenesorbitan monooleate (TWEEN-80).16-25. (canceled)
 26. A method for protecting a recombinant AAV virionfrom loss of activity resulting from storage of the virion in a glassvessel comprising admixing the virion with a virion-stabilizingcomposition comprising a dihydric or polyhydric alcohol.
 27. The methodof claim 26, wherein the dihydric or polyhydric alcohol is one or morealcohols selected from the group consisting of polyethylene glycol,propylene glycol and sorbitol.
 28. The method of claim 27, wherein theone or more alcohols is sorbitol.
 29. The method of claim 27, whereinthe one or more alcohols is polyethylene glycol.
 30. The method of claim27, wherein the one or more alcohols is propylene glycol.
 31. The methodof claim 26, wherein the virion-stabilizing composition furthercomprises a detergent.
 32. The method of claim 31, wherein the detergentis a sorbitan ester.
 33. The method of claim 32, wherein the sorbitanester is selected from the group consisting of polyoxyethylenesorbitanmonolaurate (TWEEN-20), polyoxyethylenesorbitan monopalmitate(TWEEN-40), polyoxyethylenesorbitan monostearate (TWEEN-60),polyoxyethylenesorbitan tristearate (TWEEN-65), polyoxyethylenesorbitanmonooleate (TWEEN-80) and polyoxyethylenesorbitan trioleate (TWEEN-85).34. The method of claim 26, wherein the recombinant AAV virion isprovided as a lyophilized preparation.
 35. A method for protecting arecombinant AAV virion from loss of activity resulting from storage ofthe virion in a glass vessel comprising admixing the virion with avirion-stabilizing composition comprising sorbitol and a sorbitan esterselected from the group consisting of polyoxyethylenesorbitanmonolaurate (TWEEN-20) and polyoxyethylenesorbitan monooleate(TWEEN-80).